Date of Award

1-1-2022

Embargo Period

4-22-2022

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Microbiology and Immunology

College

College of Graduate Studies

First Advisor

Stephen Tomlinson

Second Advisor

Ramin Eskandari

Third Advisor

Silvia Guglietta

Fourth Advisor

Onder Albayram

Fifth Advisor

Azizul Haque

Abstract

Germinal Matrix Hemorrhage (GMH) is a devastating neurologic pathology in neonates that leads to impaired neurodevelopmental processes and approximately 90% rate of morbidity and mortality within two years in severe cases. Recent studies in our lab have indicated a role for complement in the initiation and propagation of secondary injury after GMH, which includes neuroinflammation and hydrocephalus development. Here we investigate a strategy to target complement inhibition specifically to sites of P-selectin expression, a relevant adhesion molecule at sites of vascular injury/inflammation, as well as investigate the role of P-selectin in leukocyte recruitment and the propagation GMH secondary injury. We prepared two fusion proteins consisting of anti-P-selectin single chain antibodies (scFv) linked to Crry, a complement inhibitor. One of the scFv targeting vehicles (2.12scFv) additionally blocked the cell adhesion site of P-selectin, whereas the other (2.3scFv) bound P-selectin without blocking its function. For our investigations, post-natal mice on day 4 (P4) were subjected to collagenase induced-GMH and treated with 2.3Psel-Crry, 2.12Psel-Crry, or vehicle. Histopathological and behavioral analyses were performed at P14 and P45. After GMH injury, 2.3Psel-Crry treatment resulted in reduced infarct size and neurological deficits with improved survival, whereas 2.12Psel-Crry treatment resulted in worse outcomes compared to vehicle. MRI analyses revealed that 2.3Psel-Crry also reduced post-hemorrhagic hydrocephalus (PHH) development. We also found that 2.12Psel-Crry, but not 2.3Psel-Crry, had an anti-coagulative effect as determined by increased bleeding time and decreased heterotypic platelet interactions. P-selectin expressed on platelets is known to have a pro-coagulative function, and this effect of 2.12Psel-Crry likely explains the worse outcomes when used to treat this hemorrhagic condition. In summary, we show that GMH induces expression of P-selectin, the targeting of which with a complement inhibitor is protective against secondary neuroinflammation and development of PHH. Although unexpected, the worsened outcomes with a targeted construct that also inhibits P-selectin function (ie. 2.12Psel-Crry) can be explained by its effect on interfering with the coagulation cascade. Whereas the 2.3Psel-Crry construct has potential for protecting against the pathogenic sequelae of GMH, the 2.12Psel-Crry construct has promising potential for treatment of conditions that incorporate pathological thrombotic events, such as ischemic stroke.

Rights

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